This invention pertains to an improved form of alginate bead substrate in which bioactive cells, such as mammalian hybridomas, are captured, which substrate is perfusable with nutrient and cell product-carrying media. This invention also pertains to methods for making such a substrate and for using such a substrate, particularly to produce and to harvest cellular products in a continuous manner.
Gelled alginate beads are known to be useful as immobilization substrates for bioactive cells of various types, including bacteria, yeast, plant cells, hybridomas and animal tissue, for culturing such cells and for harvesting products thereof. This technology, with specific references to animal cells, is disclosed generally, for example, by C. Bucke (Cell Immobilization in Calcium Alginate, Methods in Enzymology, Vol. 135, pp. 175-189 (Academic Press, Inc. 1989)) and G. A. King et al., (Alginate-Polylysine Microcapsules of Controlled Membrane Molecular Weight Cutoff for Mammalian Cell Culture Engineering (Biotechnology Progress, Vol 3, No. 4, Dec. 1987, pp. 231-240)). A discussion of the specific application of such immobilization technology to the capture and culturing of animal cells has also been described by K. Nilsson (Entrapment of Cultured Cells in Agarose Beads, Large-Scale Cell Culture Technology, pp. 96-111 (Hanser Publishers, Distributed by MacMillan Publishing Company)).
Specific processes and materials for immobilizing and using animal cells in alginate gel beads has been the subject of research by the Hoffmann-LaRoche Company, of Nutley, N.J. 07110, Damon Biotech of Needham Heights, Mass. 02194, Karyon Technology, Inc. (subsequently acquired by the Schering Plough Company) and the University of Michigan, which research is believed to be reflected respectively in the following publications:
1. Techniques for Mammalian Cell Immobilization, Familletti and Fredericks, Bio/Technology, Vol. 6, January 1988, pp. 41-44. PA1 2. Microencapsulation Technology for Large-Scale Antibody Production, Posillico, Bio/Technology, Vol. 4, February 1986, pp. 114-117. PA1 3a. Karyon Technology News, September 1984; PA1 3b. Karyon Technology News, February 1985; PA1 3c. U.S. Pat. No. 4,778,749--Frye et al. PA1 4. New Macroencapsulation Technique Developed, Pobojewski, Genetic Engineering News, November/December 1988, pp. 1 and 61.
As indicated in the September 1984, Karyon Technology News, supra, animal cell growth occurs when cells are entrapped in alginate beads which are then placed in a standard tissue culture media. Cell proliferation is made possible as a result "of the high porosity of the beads allowing for diffusion of nutrients and waste products into and out of the gel matrix." U.S. Pat. No. 4,778,749 also indicates that the porosity of the alginate gel beads, in which the cellular material is immobilized, is important, but does not indicate the extent of this porosity or how it is produced.
In the Damon Biotech procedure (reference 2 above), cells are encapsulated in a two-stage process. Cells are first immobilized in a gelled sodium alginate sphere. These spheres are then coated with a biopolymer, more specifically, a semi-permeable membrane layered onto the periphery of the gelled sphere by step-wise addition of reagents including a polycationic polyamino acid compound that binds to the alginate (polyanion) spheres through salt-bond formation. When the capsule is complete, brief exposure to a chelating agent reliquifies the intracapsular alginate, allowing cells to migrate within the capsule and facilitating diffusion of nutrient medium into the capsules. Cells are protected within the capsules, and large biomolecules, (greater than 60,000 molecular weight) secreted by cells trapped in the capsular space, are trapped until harvesting.
In the University of Michigan process (reference 4 above), cells to be cultivated are mixed in a dilute calcium chloride solution which is then added dropwise to an alginate solution and forms capsules. The gelled alginate is said to remain on the outside of the capsule while calcium fuses to the surface and forms a thin skin, enabling researchers to tailor the capsule to a given thickness and size with specific characteristics.
The Hoffmann-LaRoche approach (see reference 1 above) is to combine alginate with solid gelatin particles and then to gel the Alginate as beads. By heating the beads to 37.degree. C., at which temperature the gelatin liquifies, cavities are formed in the alginate matrix. Upon culturing, cells originally disposed in the alginate suspension grow into the void space created by liquification of the gelatin.
Bead size in this procedure is said to be 8 mm in diameter. Similar work by the present inventor confirms that, because of the large solid particle size of gelatin, this method cannot be used for the production of small beads. Large particle size alginate beads exhibit relatively poor mass transport of nutrients and cellular products to the cells in the core of the bead.
Notwithstanding the foregoing background work and efforts of others, there remains a need for an improved substrate capable of immobilizing bioactive cells, such as mammalian cells on the order of 15 .mu. in diameter, which substrate permits ready accessibility to the immobilized cells, of nutrient and/or cellular product-carrying media.